Process for the preparation of pure 6-β-halopenicillanic acids and salts thereof

ABSTRACT

This invention relates to penicillanic acid derivatives of the formula I ##STR1## in which X stands for chlorine, bromine or iodine, to pharmaceutically acceptable, non-toxic salts of the compounds of formula I, to pharmaceutically acceptable, easily hydrolyzable esters thereof, including salts of such esters, to pharmaceutical compositions containing the compounds of the invention and dosage units thereof, to methods for the preparation of the compounds of the invention, and to methods of using the said new compounds in the human and veterinary therapy. 
     The 6β-halopenicillanic acids of formula I are potent inhibitors of β-lactamases from a variety of gram-positive and gram-negative bacteria, making the 6β-halopenicillanic acids as well as their salts and easily hydrolyzable esters valuable in human and veterinary medicine.

This is a continuation of application Ser. No. 564,901, filed Dec. 23,1983, now U.S. Pat. No. 4,594,246, which is a continuation of Ser. No.207,614 filed Nov. 17, 1980, now U.S. Pat. No. 4,446,144, which is acontinuation-in-part of Ser. No. 145,880, filed May 1, 1980, now U.S.Pat. No. 4,511,512.

The present invention relates to penicillanic acid derivatives of theformula I ##STR2## in which X stands for chlorine, bromine or iodine, topharmaceutically acceptable, non-toxic salts of the compounds of formulaI, to pharmaceutically acceptable, easily hydrolyzable esters thereof,including salts of such esters, to pharmaceutical compositionscontaining the compounds of the invention and dosage units thereof, tomethods for the preparation of the compounds of the invention, and tomethods of using the said new compounds in human and veterinary therapy.

In the treatment of bacterial infections it is a serious problem thatβ-lactamase producing bacteria are occurring with increasing frequency.These enzymes inactivate a variety of β-lactam antibiotics, and it iswell recognized that β-lactamases from both gram-positive andgram-negative bacteria contribute significantly to the resistance ofbacteria to β-lactam antibiotics.

It has now been found that the 6β-halopenicillanic acids of formula Iare potent inhibitors of β-lactamases from a variety of gram-positiveand gram-negative bacteria. This property makes the 6β-halopenicillanicacids as well as their salts and easily hydrolyzable esters valuable inhuman and veterinary medicine because they can protect β-lactamantibiotics against inactivation when co-administered with these.

In addition to the inhibitory activity against β-lactamases, the6β-halopenicillanic acids have antibacterial properties (cf. Table I)and are particularly active against Neisseria species.

Apart from 6β-bromopenicillanic acid, which has been mentioned in theliterature (see below), the compounds of the invention are new, and noneof them, including 6β-bromopenicillanic acid, has hitherto been obtainedin a pure state.

It has been reported (J. Org. Chem. Vol. 43, pp. 3611-3613, 1978; Proc.Natl. Acad. Sci. USA, Vol. 75, pp. 4145-4149, 1978; U.S. Pat. No.4,180,506 (Dec. 25, 1979); Biochem. J., Vol. 177, pp. 365-367, 1979)that mixtures of 6β- and 6α-bromopenicillanic acids are obtained eitheron epimerization of the latter or by selective hydrogenation of6,6-dibromopenicillanic acid, the 6β-bromo epimer being present in thereaction mixtures in estimated amounts of from 5 to 15%. The sameliterature has reported that such epimeric mixtures act as inhibitors ofβ-lactamases, and since pure 6α-bromopenicillanic acid has no effect onthese enzymes, the inhibitory activity has been attributed to the6β-bromo isomer. After the submission of the priority documentcorresponding to the present application, it has been reported(Tetrahedron Letters No. 48, pp. 4631-4634, Nov. 1979) that selectivereduction of trimethylsilyl 6,6-dibromopenicillanate with tri-n-butyltinhydride followed by hydrolysis and salt formation afforded a 30 percentyield of sodium 6β-bromopenicillanate containing less than 5 percent ofthe 6α-bromo epimer, but that the major side reaction was overreductionto penicillanic acid. The same literature also describes a similarreduction of the corresponding 6-chloro-6-iodopenicillanate providing a39 percent yield of a mixture of 6β- and 6α-chloropenicillanic acidscontaining about 25 percent of the 6α-epimer. However, neither 6β-bromo-and 6β-chloropenicillanic acid nor salts and easily hydrolyzable estersof these compounds have so far been isolated in a pure crystallinestate. 6β-iodopenicillanic acid or its salts and esters have not beenreported previously in the literature.

Thus, it is one object of the present invention to provide6β-halopenicillanic acids of formula I and salts thereof in anessentially pure, crystalline form, suitable for medical use, thesecompounds exhibiting strong β-lactamase inhibitory activity increasingin the order Cl<Br<I and also showing antibacterial activity, inparticular against Neisseria species.

Another object of the present invention is to provide easilyhydrolyzable esters of the acids of formula I and salts of such estersbeing useful as pro-drugs and/or intermediates.

It is a further object of the invention to provide combinations of thecompounds of the invention with selected β-lactam antibiotics, withwhich the new compounds act synergistically against a variety ofβ-lactamase producing bacteria because they protect the β-lactamantibiotics against inactivation by these enzymes.

Other objectives of the present invention will be apparent from thefollowing description.

The salts of the 6β-halopenicillanic acids are salts withpharmaceutically acceptable, non-toxic bases, and among the suitablesalts mention may be made of alkali metal salts and alkaline earth metalsalts, such as lithium, sodium, potassium, magnesium, and calcium salts,as well as salts with ammonia and suitable non-toxic amines, such aslower alkylamines, e.g. triethylamine, lower alkanolamines, e.g.diethanolamine or triethanolamine, procaine, cycloalkylamines, e.g.dicyclohexylamine, benzylamines, e.g. N-methylbenzylamine,N-ethylbenzylamine, N-benzyl-β-phenethylamine,N,N'-dibenzylethylenediamine or dibenzylamide, and heterocyclic amines,e.g. morpholine, N-ethylpiperidine or the like. Also salts formed withe.g. β-lactam antibiotics or pro-drugs thereof containing a basic group,such as pivampicillin, pivmecillinam, bacampicillin, bacmecillinam,penethamate, ampicillin or amoxycillin, are within the scope of theinvention. It has been surprisingly found that the last mentioned saltsare readily crystallized and are therefore particularly suitable asagents in pharmaceutical preparations giving rise in the body of thepatient to a simultaneous presence of the β-lactam antibiotic inquestion and the β-lactamase inhibitor. In some instances, it ispreferred to use salts which are readily soluble in water, whereas forother purposes, it may be appropriate to use an only slightly solublesalt, e.g. in order to obtain a prolonged effect or for preparation ofaqueous suspensions.

The above list shall, however, only be considered illustrative for andnot limiting the present invention.

The present esters of the 6β-halopenicillanic acids are esters which arereadily hydrolyzed in vivo or in vitro. Such esters includeacyloxyalkyl, alkoxycarbonyloxyalkyl or aminoacyloxyalkyl esters of theformula II ##STR3## in which X has the same meaning as in formula I, R₁is hydrogen, methyl or ethyl, and R₂ is a straight or branched alkyl oralkoxy with from 1 to 6 carbon atoms, or an aryl or aryloxy radical, orR₂ is a straight or branched aminoalkyl radical with from 1 to 6 carbonatoms, the alkyl moiety optionally being substituted by one or moreadditional groups, such as hydroxy, mercapto, alkoxy, alkylthio,carbalkoxy, carboxamido, phenyl or hydroxyphenyl. The asterisk in theester moiety indicates the possibility of an asymmetric carbon atom.

Among the above esters the following are preferred: alkanoyloxymethylwith from 3 to 8 carbon atoms, 1-(alkanoyloxy)ethyl with from 4 to 9carbon atoms, alkoxycarbonyloxymethyl with from 3 to 6 carbon atoms,1-(alkoxycarbonyloxy)ethyl with from 4 to 7 carbon atoms, andα-aminoalkanoyloxymethyl with from 2 to 6 carbon atoms.

Other preferred esters are lactonyl esters, e.g. 3-phthalidyl,4-crotonolactonyl or γ-butyrolacton-4-yl esters.

Also within the scope of the invention are methoxymethyl, cyanomethyl,or mono- or dialkyl substituted aminoalkyl esters, e.g.2-dimethylaminoethyl, 2-diethylaminoethyl, or 3-dimethylaminopropylesters.

In particular, such esters are preferred which are well-absorbed uponoral administration and during or after the absorption are hydrolysed tothe free acids of formula I.

Esters which contain an amino group in the ester moiety can be preparedand used in the form of their salts with pharmaceutically acceptable,non-toxic inorganic or organic acids. Examples of suitable inorganic andorganic acids include, but are not limited to, the following:hydrohalide acids, e.g. hydrochloric, hydrobromic and hydriodic acid,phosphoric acid, sulphuric acid, nitric acid, p-toluenesulphonic acid,methanesulphonic acid, formic acid, acetic acid, propionic acid, citricacid, tartaric acid, maleic acid, pamoic acid,p-(dipropylsulfamyl)benzoic acid (probenecid), andphenoxymethylpenicillin or other acidic β-lactam antibiotics. Asmentioned above, easily soluble or only slightly soluble salts may bereferred for different purposes.

The present invention also relates to methods for the preparation of thecompounds of the invention including their separation from mixturescontaining the corresponding 6α-epimers and/or 6,6-dihalo derivativeswhich may be present in the crude reaction mixtures.

According to one method, mixtures of epimeric 6-halopenicillanic acidsare produced by aqueous equilibration of a salt of 6α-halopenicillanicacid at 30°-40° C. and a moderately basic pH-value (8-10) for 6-48hours, preferably at 30°-32° C. and pH 9.0-9.1 for 20-24 hours, the pHin the reaction mixture being held constant by addition of diluteaqueous base via an automatic titrator. The amount of 6β-halo epimerpresent in the resulting mixtures decreases in the order I>Br>Cl, butthe yields of the β-epimers are at least twice as high as thosedescribed in the literature for the epimerization of6α-bromopenicillanic acid or by using said method for the epimerizationof the corresponding 6α-chloro and 6α-iodo acids.

The epimeric mixtures of 6-chloro-, 6-bromo-, or 6-iodopenicillanicacids thus obtained can be separated by column chromatography on silicagel using as developing solvent an appropriate mixture of organicsolvents, e.g. ether-petroleum ether, ethyl acetate-petroleum ether,chloroform-benzene or ethyl acetate-cyclohexane, these solvent mixturespreferably containing a low percentage (0.1-0.5%) of a carboxylic acid,such as formic acid or acetic acid. A highly efficient developingsolvent for the separation of the epimeric mixtures referred to above bydry column chromatography on silica gel is e.g. ether-petroleumether-formic acid, 70:30:0.1. Hereby the more polar 6β-halopenicillanicacids are completely separated from their less polar 6α-epimers and,following a usual work-up procedure of the eluates, obtained in a purecrystalline state, either in the form of the free acids or as thecorresponding potassium or sodium salts. The purity of the crystalline6β-halopenicillanic acids thus obtained as well as the respectivepotassium and sodium salts is at least 99 percent, as determined bythin-layer and gas-liquid chromatography.

In another embodiment of the method 6,6-dihalopenicillanic acids orsalts thereof can be selectively reduced by treatment with alkali metalor tetraalkylammonium boranates, e.g. sodium borohydride, potassiumborohydride, sodium cyanoborohydride, tetrabutylammonium boranate, orcetyl trimethylammonium boranate to afford favorably high yields (>50%)of the free 6β-halopenicillanic acids of formula I. The reactions areperformed in an appropriate organic solvent, e.g. dimethyl sulphoxide,dimethylformamide, ethyl acetate or methylene chloride, and attemperatures between 0° and 80° C., preferably at room temperature. The6β-halopenicillanic acids of formula I can be separated from thecorresponding 6α-halo- and/or 6,6-dihalopenicillanic acids present inthe crude reaction mixtures by column chromatography as referred toabove or by fractionate crystallization known to the man skilled in theart.

According to a further aspect of the present invention, the new estersof the 6β-halo acids of formula I can be prepared by epimerization ofthe corresponding 6α-halopenicillanic acid esters in an appropriateorganic solvent, e.g. methylene chloride, chloroform ordimethylformamide, in the presence of an organic base, e.g.1,5-diazabicyclo[4.3.0]non-5-ene or triethylamine, and at temperaturesbetween -10° C. and room temperature. The epimeric mixtures of thecorresponding 6-halopenicillanic acid esters thus obtained are separatedby column chromatography under similar conditions as mentioned above toafford the pure 6β-halo isomers.

In a further embodiment of the method, the esters of the presentinvention can be obtained by selective reduction of6,6-dihalopenicillanic acids esters with alkali metal ortetraalkylammonium boranates, similar to the procedure referred tobefore. The 6β-halo esters thus obtained are separated from minoramounts of the corresponding 6α-epimers and/or unreacted startingmaterials by column chromatography as described above.

The 6β-halopenicillanic acids of formula I or their salts can beconverted into the corresponding esters by well known esterificationprocesses, and vice versa. Such esters can be cleaved chemically orenzymatically to give the corresponding free acids of formula I or saltsthereof under conditions which do not result in any appreciabledestruction of the remaining part of the molecule.

As it has been previously stated, the 6β-halopenicillanic acids of thepresent invention are potentiators of β-lactamase sensitive antibioticsand may by themselves be useful in combatting some specific bacterialinfections.

More specifically, the antibacterial spectra of the pure6β-halopenicillanic acids of the invention will appear from Table Ibelow.

                  TABLE I    ______________________________________    Antibacterial spectra.sup.a of 6β-bromopenicillanic acid (A),    6β-    chloropenicillanic acid (B) and 6β-iodopenicillanic acid (C)                   IC.sub.50 (ug/ml)    Organism         A         B       C    ______________________________________    Staph. aureus CJ9                     32        40      63    Dipl. pneumoniae EA                     1.6       5.0     5.0    Strep. pyogenes EC                     6.3       6.3     5.0    Strep. faecalis EI3                     >100      >100    >100    Coryneb. xerosis FF                     5.0       5.0     16    Bacillus subt. KA2                     5.0       7.9     13    Pseud. aeruginosa BA2                     >100      >100    >100    Alcaligenes faecalis GA                     2.0       2.5     10    Escherichia coli HA2                     50        50      >100    Escherichia coli HA58(RTEM)                     100       >100    >100    Kleb. pneumoniae HE                     63        63      >100    Enterobact. aerogenes HC7A                     63        63      >100    Proteus vulg. HJ 40        40      100    Salm. typhimurium HL2                     100       100     >100    Shigella dysenteriae HR                     50        40      >100    Neisseria gonorrhoeae DA2                     0.25      0.20    0.79    Neisseria meningitidis DB                     0.79      0.63    1.6    Haemophilus influenzae IX3                     32        20      >100    ______________________________________     .sup.a Determined by serial dilutions in fluid medium, inoculum 10.sup.6     CFU.sup.b (grampositive organism) or 10.sup.4 CFU (gramnegative organism)     .sup.b CFU = colony forming units

In Table II is shown the in vitro activity against β-lactamase producingbacterial strains of selected β-lactam antibiotics in 1:1 combinationswith the 6β-halopenicillanic acids of formula I. These data indicatethat, in combination with the 6β-halopenicillanic acids,benzylpenicillin and ampicillin are highly active against otherwiseresistant strains of Staphylococcus aureus. A similar synergistic effectagainst strains of Klebsiella pneumoniae, Proteus mirabilis, andEscherichia coli is shown by combinations of ampicillin as well asmecillinam with the 6β-halo acids of the invention.

                                      TABLE II    __________________________________________________________________________    Susceptibility.sup.(a) of β-lactamase producing bacterial strains to    selected β-lactam antibio-    tics in 1:1 combinations with 6β-bromopenicillanic acid (A),    6β-chloropenicillanic acid (B),    and 6β-iodopenicillanic acid (C)               IC.sub.50 (μg/ml)               Staphylococcus                       Staphylococcus                               Klebsiella                                     Proteus                                           Escherischia               aureus  aureus  pneumoniae                                     mirabilis                                           coli    Antibiotic CJ9     CJ145   HE7   HJ28  HA58 (RTEM)    __________________________________________________________________________    A          13      10      >50   >50   >50    B          16      13      >50   >50   >50    C          20      6.3     >100  >100  >100    Benzylpenicillin               >100    10      >100  >100  >100    Benzylpenicillin + A               0.5 + 0.5                       0.2 + 0.2                                ND.sup.(b)                                     ND    ND    Benzylpenicillin + B               1.3 + 1.3                       0.4 + 0.4                               ND    ND    ND    Benzylpenicillin + C               0.79 + 0.79                       0.08 + 0.08                               7.9 + 7.9                                     2.5 + 2.5                                           7.9 + 7.9    Ampicillin 10      1.6     >50   >50   >100    Ampicillin +  A               0.4 + 0.4                       0.16 + 0.16                               1.3 + 1.3                                     5.0 + 5.0                                           4.0 + 4.0    Ampicillin + B               1.6 + 1.6                       0.32 + 0.32                               ND    ND    ND    Ampicillin + C               0.79 + 0.79                       0.16 + 0.16                               2.5 + 2.5                                     2.5 + 2.5                                           3.2 + 3.2    Mecillinam >50     >50     >25   6.3   1.3    Mecillinam + A               4.0 + 4.0                       3.2 + 3.2                               5.0 + 5.0                                     1.3 + 1.3                                           0.16 + 0.16    Mecillinam + B               ND      4.0 + 4.0                               ND    ND     0.05 + 0.05.sup.(c)    Mecillinam + C               10 + 10 3.2 + 3.2                               0.79 + 0.79                                     0.63 + 0.63                                           0.13 + 0.13    __________________________________________________________________________     .sup.(a) Serial dilutions in agar modium, inoculum 10.sup.5 CFU     (grampositive organisms) or 10.sup.4 CFU (gramnegative organisms)     .sup.(b) ND = not determined     .sup.(c) corresponding to an IC of 0.4 μg/ml for mecillinam in this     test

It is also an object of the present invention to provide pharmaceuticalcompositions for use in the treatment of infectious diseases whichcontain as an active ingredient at least one of the compounds of theinvention.

The compositions include forms adapted for enteral, parenteral,intramammal or topical use and may be used for the treatment ofinfections in mammals including humans.

The free 6β-halopenicillanic acids of formula I or their salts may beused for enteral, parenteral and topical administration. However, fororal use it may in some instances be advantageous to use an easilyhydrolyzable ester of the invention or a salt thereof.

Injectable or infusable compositions of the 6β-halopenicillanic acids offormula I or their salts are suitable, when high tissue levels of the6β-halopenicillanic acids are rapidly desired, or when used incombination with a parenterally administered β-lactam antibiotic, asdescribed below.

For intramammal use it is preferred to use an ester of the inventionwhich provides an adequate local concentration, e.g. a dialkylaminoalkylester or a salt thereof.

The active ingredient can be used as such or can be mixed up withcarriers and/or auxiliary agents.

In such compositions, the proportion of therapeutically active materialto carriers and auxiliary agents can vary between 1% and 95%. Thecompositions can be worked up to pharmaceutical forms of presentationsuch as tablets, capsules, powders, syrups, suspensions, solutions,including forms suitable for injection or infusion.

The carriers and/or auxiliary agents are pharmaceutically acceptablematerials such as gelatine, lactose, starch, magnesium stearate, talc,vegetable and animal fats and oils, gum, polyalkylene, glycol, or otherknown carriers for medicaments, and diluents, binders, buffers,preservatives, disintegrants, coating materials, and the like inaccordance with pharmaceutical practice in the manner well understood bythose skilled in the art, in order to provide appropriate forms ofpharmaceutical presentation, including sustained release preparations,double tablets containing the therapeutically active ingredientsseparated from each other, and enteric coated tablets, etc.

The compounds of the invention may be present in the composition as thesole agent or together with other therapeutic agents, in particular aβ-lactam antibiotic or a synergistic combination of β-lactamantibiotics. Suitable β-lactam antibiotics for such compositions includenot only those known to be highly susceptible to β-lactamases, but alsothose which have a good degree of intrinsic resistance to β-lactamases.Thus, suitable β-lactam antibiotics for such compositions includebenzylpenicillin, phenoxymethylpenicillin, carbenicillin, methicillin,propicillin, ampicillin, amoxycillin, epicillin, ticarcillin,cyclacillin, cephaloridine, cephalothin, cefazolin, cephalexin,cefaclor, cephacetrile, cephamandole, cephapirin, cephradine,cephaloglycine, mecillinam, and other well known penicillins,cephalosporins or amidinopenicillanic acids or pro-drugs thereof, suchas hetacillin, metampicillin, the acetoxymethyl, pivaloyloxymethyl,ethoxycarbonyloxyethyl, and phthalidyl esters of benzylpenicillin,ampicillin, amoxycillin or cephaloglycine, or the phenyl, tolyl, andindanyl α-esters of carbenicillin, ticarcillin or the like, or 6β-amidinopenicillanic acid pro-drugs, like pivmecillinam orbacmecillinam, or a similar 7β-amidinocephalosporanic acid derivative.

When present in a pharmaceutical composition together with anotherβ-lactam antibiotic, the ratio of the compounds of the invention to theother β-lactam antibiotic(s) is from 10:1 to 1:10 and advantageously maybe from 3:1 to 1:3, calculated as the free acids, the range, however,not to be considered limiting the invention.

Pharmaceutical preparations for intramammary use and similar localtreatment of cavities are of particular interest. It has been found that6-β-halo penicillanic acid administered as a salt or an easilyhydrolyzable ester rapidly penetrates the udder tissue causing in serumand milk a high concentration of the 6-β-halo penicillanic acid which inturn causes a protection of a β-lactam antibiotic used in the treatmentof an infection and/or enchances the effect of the antibiotic inquestion. Specifically advantageous pharmaceutical preparations of theinvention consequently comprise anti-mastitis preparations containingbesides the antimicrobial agents, in particular, β-lactam antibiotics,as such or as pro-drugs, 6-β-halo penicillanic acid in the form of oneor more of its salts or easily hydrolyzable esters, or salts of suchesters. Such salts and esters can according to the invention alsoconsist of β-lactam antibiotic or other antibiotics used as salt orester component. Among suitable β-lactam antibiotics for suchantimastitis preparations can be mentioned: benzylpenicillin,ampicillin, amoxycillin, carbenicillin, cloxacillin, flucloxacillin,ticarcillin, nafcillin, dicloxacillin, oxacillin, methicillin,carfecillin, mecillinam, cephaloridine, cephalexin, cephacetrile andmixtures of one or more of such β-lactam antibiotics, for example;ampicillin/cloxacillin, amoxycillin/cloxacillin,ampicillin/flucloxacillin, amoxycillin/flucloxacillin,ticarcillin/flucloxacillin and ampicillin/mecillinam, without thisenumeration being considered as limiting.

A pharmaceutical preparation which is specifically of importancecontains as the antibiotic part benzylpenicillin in the form of itsβ-diethylaminoethyl ester having the generic name, penethamate.

Penethamate as such or in the form of its hydrochloride is known to bevery effective in the treatment of mastitis by giving rise to a rapidand high concentration of benzylpenicillin in the udder.

As is well known benzylpenicillin is very sensitive to β-lactamases andconsequently the combination of penethamate and 6-β-halo penicillanicacid in an anti-mastitis preparation is appropriate due to itsprotection of benzylpenicillin. In such a preparation other β-lactamantibiotics, e.g., those mentioned above, have been included in order toobtain a broader antibacterial spectrum and/or an enhancement of theantibacterial activity and/or a synergistic effect. In a particularembodiment the penethamate salt of 6-β-halo penicillanic acid can beused in the combination.

Other antibiotics which can be present in the anti-mastitis preparationaccording to the invention are by way of example: streptomycin,dihydrostreptomycin, neomycin, polymyxin B and E, tetracyclines,framycetin, colimycin, gentamycin, erythromycin, canamycin, bacitracin,tyrothricin and sulfa drugs such as sulfamerazine, sulfathiazole orsulfanilamide.

Appropriately, and in accordance with common practice, the anti-mastitispreparation can also contain, for example, a corticosteroid orpain-relieving agents or other similar acting components.

The composition of the invention for use in the treatment of mastitiscomprises suspensions or solutions of the active components in asuitable vehicle which can be made of an aqueous or oily base.

The composition can be administered in conventional ways for thetreatment of mastitis, e.g., with an intramammary injector or in theform of an aerosol. In the preferred embodiment the composition can beadministered as an aerosol foam.

With a view to obtaining the highest degree of stability it is preferredas a vehicle to apply non-aqueous media such as vegetable oils orparaffin oil. Appropriately a thickening agent can be added such as12-hydroxystearin or aluminum/stearate. Also as thickening agents therecan be used beeswax, hydrogenated peanuts or castor oil or soft or hardparaffin. Furthermore, as a thickener use can be made of lactose orcastor oil with modified viscosity after treatment with ethyleneoxideand marketed under the trade name "Cremophor".

Further, the composition used in the treatment of mastitis canappropriately contain detergents such as "Span" (fatty acid sorbate) or"Tween" (fatty acid poly sorbate).

Conventionally such additives are used in amounts up to 10 w/v percentof the composition, whereas the amount of detergent appropriately variesbetween 0.1 and 8 w/v percent.

The vegetable oil can advantageously be chosen from among commerciallyavailable oils, the fatty acid of which contains from 8 to 10 carbonatoms. Particularly useful is the commercial product "Viscoleo" which ishydrogenated castor oil with low viscosity.

The amount of the therapeutically active agents in total in theanti-mastitis preparation above can appropriately vary from 0.5 to 20w/v percent.

Another object of the invention resides in the selection of a dose ofthe compounds of the invention and a dosage unit of the compositions ofthe invention which dose and dosage unit can be administered so that thedesired results are achieved without simultaneous secondary effects.

The compositions of the invention are conveniently administered indosage units containing a total amount of from 0.025 g to 2.5 g, andpreferably from 0.1 g to 1.0 g, of the antibacterial agents, calculatedas the free acids. The expression "antibacterial agents" shall here andin the following mean one or more compounds of the invention, alone orcombined with one or more known β-lactam antibiotics, salts or pro-drugsthereof. When used in the veterinary practice, the dosage units maycontain up to 25 g of the antibacterial agents.

By the term "dosage unit" is meant a unitary, e.g. a single dose capableof being administered to a patient, and which may be readily handled andpacked, remaining as a physically stable unit dose, comprising eitherthe active materials as such or a mixture thereof with a pharmaceuticalcarrier.

Similarly, for infusion, the compositions of the invention are given indosage units containing up to 10 g of the antibacterial agents inaqueous solution.

For parenteral use, e.g. injections, the compositions of the inventionare given e.g. in an aqueous solution or suspension as a dosage unitcontaining from 0.1 g to 1 g of the antibacterial agents, calculated asthe free acids, to be dissolved or suspended immediately before use, orready for use together with a pharmaceutically acceptable vehicle.

In the form of a dosage unit the compounds may be administered once ormore times a day at appropriate intervals, always depending, however, onthe condition of the patient.

Thus, a daily dose will amount to from 0.1 g to 30 g (corresponding to1-425 mg/kg body weight/day), preferably from 0.2 g to 6 g of theantibacterial agents, calculated as the free acids.

The compositions of the invention may be used in the treatment ofinfections of, inter alia, the respiratory tract, the urinary tract, andsoft tissues in humans and may also be used to treat infections inanimals such as mastitis in cattle.

In compounded compositions containing other β-lactam antibiotics, thelatter will normally be present in approximately the same amounts asconventionally used when such β-lactam antibiotics are the soletherapeutic agents, but under certain circumstances it may beappropriate to reduce the amounts thereof.

Particularly favored compounded compositions will contain from 50-1000mg of the β-lactam antibiotic, a salt or a pro-drug thereof, and the6β-halopenicillanic acid, a salt or a pro-drug thereof, in an amountwithin the aforementioned ratios, and more suitably from 200-500 mg ofthe β lactam antibiotic, a salt or a pro-drug thereof, and from 25-250mg of the 6β-halopenicillanic acid, a salt or a pro-drug thereof.

The compounds of the invention may be administered in the form of theirpharmaceutically acceptable, non-toxic esters. The term "non-toxic" forsuch esters means that they are therapeutically acceptable for theirintended form of administration. In general, the esters of the compoundsof the invention are used in the oral administration, but their use inthe parenteral administration is also within the scope of the invention.

The present invention also comprises a method for the treatment ofhumans and other mammals, consisting in the administration of aneffective amount of the present compositions to patients suffering frominfectious diseases.

In the treatment of mastitis the composition or compounded compositionof the invention is appropriately administered by an injector(intramammary) containing e.g. 5 ml.

In the lactation period in which a short period of treatment isdesirable, the composition is appropriately given a dose amounting to5-10 ml per quarter of a composition with a base consisting mostly ofvegetable oils in order to obtain rapid excretion of the β-lactamantibiotics.

In the dry period the composition or compounded composition canappropriately be based upon paraffin oils with aluminum stearate andpreferably containing other antimicrobial agents, e.g. streptomycin.

The method may consist in administering compositions or compoundedcompositions of the invention, or in administering such compositionscontaining the compounds of the invention alone together withcompositions containing other β-lactam antibiotics. In the latter case,the two types of compositions may be administered simultaneously or atintervals and with varying proportions between the 6β-halopenicillanicacid and the β-lactam antibiotic.

According to the invention between 0.1 and 30 g of the antibacterialagents will be administered each day of the treatment, but more oftenbetween 500 and 6000 mg of the antibacterial agents will be administeredper day.

In the treatment of mastitis the infected quarter is treated withcontents of the intramammary every 24 hours in the lactation perioduntil the infection ceases, normally after 3 treatments, and in the dryperiod the infected quarter is appropriately treated with a singlelong-term dose.

It shall be expressly understood that the above ranges of doses indicatethe total amount of antibacterial agents, i.e. one or more compounds ofthe invention administered either alone, combined with, or given atintervals with other β-lactam antibiotics as above.

The drawings (FIG. 1 and FIG. 1a) represent NMR-spectra referred to inExample 1.

The invention will be further described in the following Examples whichare not to be construed as limiting the invention.

EXAMPLE 1 Potassium 6β-bromopenicillanate

A solution of potassium 6α-bromopenicillanate (7.64 g, 24 mmol) in 0.04Maqueous disodium hydrogen phosphate (800 ml) was incubated for 72 hoursat 30° C. According to an NMR-spectrum (D₂ O) of a freeze-dried 5 mlsample, the epimeric mixture contained 10-20% of the 6β-bromo compound.

After addition of sodium chloride (160 g), the mixture was stirred at 0°C. under a layer of ether (250 ml), and the pH of the aqueous phase wasadjusted to 3 with 4N aqueous hydrochloride. The organic layer wasseparated, the aqueous phase was re-extracted with ether (100 ml), andthe combined ethereal extracts were washed with saturated aqueous sodiumchloride (10 ml), dried, and concentrated to about 40 ml at reducedpressure. The concentrated solution was subjected to dry columnchromatography on silica gel (Silica Woelm TSC, Woelm Pharma, Eschwege,Western Germany). The column (φ5.6 cm, length 46 cm) was developed withether-petroleum ether-formic acid, 70:30:0.1 (1200 ml), fractions a 2 cmwere scraped out, suspended in ethyl acetate (10 ml/fraction), andsamples of the supernatants were examined by thin-layer chromatographyusing the above mentioned solvent system. Fractions containing the pure,more polar 6β-bromopenicillanic acid were combined and eluted with ethylacetate. The resulting ethyl acetate eluate was concentrated to about 50ml at reduced pressure and washed thoroughly with water (6×5 ml) toremove the major amount of formic acid. To the organic layer was addedwater (40 ml), and the apparent pH of the mixture was adjusted to 7.2 byaddition of 0.5M aqueous potassium bicarbonate. The aqueous layer wasseparated and freeze-dried to afford 0.54 g of pure potassium6β-bromopenicillanate as a colorless amorphous powder which crystallizedfrom n-butanol, [α]_(D) ²⁰ +240° (c=0.2, H₂ O).

The detailed FT proton NMR-spectrum (FIG. 1) showed signals at δ=1.47(s, 3H; CH₃ -2α), 1.59 (s, 3H; CH₃ -2β), 4.27 (s, 1H; CH-3), 5.52 and5.58 (doublets, J=4 Hz, 2H; CH-5α and CH-6α, confer FIG. 1a) ppm.

Instrument JEOL FX 100. Concentration 50 mg per ml. All data convertedto tetramethylsilane as 0.00 ppm δ-scale.

EXAMPLE 2 Potassium 6β-chloropenicillanate

A solution of potassium 6α-chloropenicillanate (13.14 g, 48 mmol) in0.04M aqueous disodium hydrogen phosphate (1600 ml) was incubated for 96hours at 30° C. to yield, as revealed by an NMR-spectrum (D₂ O) of afreeze-dried 5 ml sample of the reaction mixture, about 5-6% of6β-chloropenicillanic acid in admixture with the starting material.

To the reaction mixture was added sodium chloride (320 g) and ether (400ml), and the pH of the aqueous phase was adjusted to 3 by addition of 4Naqueous hydrochloric acid at 0° C. with stirring. The organic phase wasseparated, the aqueous layer was re-extracted with ether (200 ml), andthe combined ethereal extracts were washed with saturated aqueous sodiumchloride (20 ml), dried, and concentrated to about 50 ml at reducedpressure. The concentrate was subjected to dry column chromatography onsilica gel (as described in Example 1 for the separation of thecorresponding 6-epimeric bromopenicillanic acids). Fractions containingthe pure 6β-chloropenicillanic acid were eluted with ethyl acetate, andthe resulting solution was worked up in a similar manner as described inExample 1 to afford 0.68 g of potassium 6β-chloropenicillanate as anamorphous powder which crystallized from n-butanol.

The NMR-spectrum (D₂ O) showed signals at δ=1.48 (s, 3H; CH₃ -2α), 1.58(s, 3H; CH₃ -2β), 4.27 (s, 1H, CH-3), 5.43 and 5.63 (2d, J=4 Hz, 2H;CH-5α and CH-6α) ppm. Tetramethylsilane was used as external reference.

EXAMPLE 3 Potassium 6β-iodopenicillanate

By following the procedure of Example 1, but substituting potassium6α-iodopenicillanate for the potassium 6α-bromopenicillanate, thedesired compound was obtained as an amorphous product which crystallizedfrom n-butanol.

EXAMPLE 4 Potassium 6β-bromopenicillanate

A solution of potassium 6α-bromopenicillanate (15.28 g, 48 mmol) inwater (320 ml) was adjusted to pH 9.0 with 1N aqueous sodium hydroxideand stirred for 24 hours at 30° C. During the reaction a pH of 9.0 wasmaintained in the solution by addition of 1N aqueous sodium hydroxidevia an automatic titrator. An NMR spectrum (D₂ O) obtained from afreeze-dried 1 ml sample of the solution indicated the presence ofapproximately 25% of the 6β-bromo compound in the epimeric mixtureformed.

The mixture was worked up and purified by column chromatography asdescribed in Example 1 to yield crystalline potassium6β-bromopenicillanate identical with the product prepared in Example 1;[α]_(D) ²⁰ +253° (c=0.5, 1M phosphate buffer, pH 7).

Calculated for C₈ H₉ BrKNO₃ S: C, 30.19; H, 2.85; Br, 25.11; N, 4.40; S,10.08%. Found: C, 30.16; H, 2.95; Br, 25.28; N, 4.33; S, 10.07%.

EXAMPLE 5 Potassium 6β-chloropenicillanate

By following the procedure of Example 4, but substituting potassium6α-chloropenicillanate for the potassium 6α-bromopenicillanate, anepimeric mixture containing about 15% of the 6β-chloro compound wasobtained, as revealed by an NMR spectrum (D₂ O) of a freeze-dried sampleof the reaction mixture.

The crystalline title compound was obtained using a similar work-up andchromatography method as described in Example 1; [α]_(D) ²⁰ +243°(c=0.5, 1M phosphate buffer pH 7).

EXAMPLE 6 Potassium 6β-iodopenicillanate A. Acetoxymethyl6-diazopenicillanate

To a stirred solution of acetoxymethyl 6β-aminopenicillanate (5.77 g, 20mmol) and sodium nitrate (2.76 g, 40 mmol) in a mixture ofdichloromethane (120 ml) and water (120 ml) was added dropwise at 0°-3°C. 4N aqueous sulphuric acid (7 ml).

After stirring at the low temperature for a further 30 minutes, theorganic phase was separated, dried (Na₂ SO₄), and concentrated toapproximately 30 ml at reduced pressure.

This concentrate was used immediately in the following step.

B. Acetoxymethyl 6α-iodopenicillanate

The concentrated solution of acetoxymethyl 6-diazopenicillanate fromstep A above was diluted with ice cold acetone (180 ml), and to thestirred mixture was added dropwise at 0°-3° C. a solution of sodiumiodide (9.0 g, 60 mmol) and 57% hydroiodic acid (7.4 ml) in water (15ml). After stirring at 0°-3° C. for a further 25 minutes, the mixturewas treated with solid sodium bicarbonate (10 g) and filtered. Thefiltrate was diluted with ethyl acetate (150 ml), acetone was removed atreduced pressure, and the remaining organic layer was separated, washedwith 0.5M aqueous sodium thiosulphate (2×100 ml), dried (Na₂ SO₄), andconcentrated to about 10 ml at reduced pressure.

This concentrated solution was subjected to dry column chromatography onsilica gel (ether-petroleum ether, 4:6) to yield pure acetoxymethyl6α-iodopenicillanate as a slightly yellowish oil.

The NMR spectrum (CDCl₃) showed signals at δ=1.48 (s, 3H; CH₃ -2α), 1.63(s, 3H; CH₃ -2β), 2.11 (s, 3H; COCH₃), 4.56 (s, 1H; CH-3), 4.99 (d,J=1.5 Hz, 1H; CH-6), 5.45 (d, J=1.5 Hz, 1H; CH-5), and 5.79 (ABq, J=5.5Hz, 2H; OCH₂ O) ppm.

Tetramethylsilane was used as internal reference.

C. Potassium 6α-iodopenicillanate

To a solution of acetoxymethyl 6α-iodopenicillanate (2.0 g, 5 mmol) in70% aqueous methanol (50 ml) was added 4N aqueous hydrochloric acid (1.5ml), and, after protection from light, the mixture was stirred at roomtemperature for 3 days. The mixture was poured into water (150 ml),extracted twice with ether (100 ml), and the combined ethereal extractswere washed with water (2×25 ml). To the organic layer was added freshwater (40 ml), and the pH in the aqueous phase was adjusted to 6.8 byaddition of 1M potassium bicarbonate with stirring. The aqueous phasewas separated and freeze-dried to give potassium 6α-iodopenicillanate asan amorphous powder, which crystallized from acetone.

The NMR-spectrum (D₂ O)) showed signals at δ=1.46 (s, 3H; CH₃ -2α), 1.57(s, 3H; CH₃ -2β), 4.30 (s, 1H; CH-3), 5.24 (d, J=1.5 Hz, 1H; CH-6), and5.46 (d, J=1.5 Hz, 1H: CH-5) ppm.

D. Potassium 6β-iodopenicillanate

A solution of potassium 6α-iodopenicillanate (3.65 g, 10 mmol) in water(200 ml) was stirred at 30° C. for 20 hours, a constant pH of 9.0 beingmaintained in the reaction mixture by additions of 0.1N sodium hydroxidevia an automatic titrator. According to the NMR spectrum (D₂ O) of afreeze-dried 1 ml-sample, the epimeric mixture of 6-iodopenicillanatesthus formed contained approximately 30% of the 6β-iodo compound.

To the mixture was added ether (150 ml), and the pH of the aqueous phasewas adjusted to 3.0 by addition of 4N hydrochloric acid with stirring.The organic phase was separated, the aqueous phase re-extracted withether (50 ml), and the combined ethereal extracts were washed withsaturated aqueous sodium chloride (2×20 ml), dried (MgSO₄), andconcentrated to about 6-8 ml at reduced pressure. The concentrate thusobtained was subjected to dry column chromatography on silica gel(ether-petroleum ether-formic acid, 70:30:0.1), and, analogously to theprocedure described in Example 1 for the separation and isolation of thecorresponding 6β- and 6α-bromo compounds, potassium 6β-iodopenicillanatewas obtained in a crystalline state; [α]_(D) ²⁰ +260° (c=0.5, 1Mphosphate buffer pH 7).

The NMR spectrum (D₂ O) showed signals at δ=1.49 (s, 3H; CH₃ -2α), 1.65(s, 3H; CHHD 3-2β), 4.29 (s, 1H; CH-3), 5.42 and 5.80 (2d, J=3.5 Hz, 2H;CH-5, and CH-6) ppm.

Calculated for C₈ H₉ IKNO₃ S: C, 26.31; H, 2.48; I, 34.75; N, 3.84; S,8.78%. Found: C, 26.51; H, 2.58; I, 34.91; N, 3.675; S, 8.80%.

EXAMPLE 7 Pivaloyloxymethyl 6β-bromopenicillanate

To a solution of potassium 6β-bromopenicillanate (0.64 g, 2 mmol) indimethylformamide (15 ml) was added chloromethyl pivalate (0.27 ml, 2.5mmol), and the mixture was stirred for 16 hours at room temperature.After dilution with ethyl acetate (45 ml), the mixture was washed withwater (4×10 ml), dried (MgSO₄), and evaporated in vacuo. The residualoil crystallized from diisopropyl ether to give the title compound,melting point: 67°-68° C.

The NMR spectrum (CDCl₃) showed signals at δ=1.23 (s, 9H; C(CH₃)₃), 1.51(s, 3H; CH₃ -2α), 1.68 (s, 3H; CH₃ -2β), 4.54 (s, 1H; CH-3), 5.32 and5.57 (2 d, J=4 Hz, 2H; CH-5 and CH-6), and 5.82 (ABq, J=5.5 Hz, 2H; OCH₂O) ppm. Tetramethylsilane was used as internal reference.

EXAMPLE 8 Pivaloyloxymethyl 6β-chloropenicillanate

By substituting potassium 6β-chloropenicillanate for the potassium6β-bromopenicillanate in the procedure of Example 7, pivaloyloxymethyl6β-chloropenicillanate was obtained as colorless crystals, meltingpoint: 68°-69° C.

The NMR spectrum (CDCl₃) showed signals at δ=1.22 (s, 9H; C(CH₃)₃), 1.51(s, 3H; CH₃ -2α), 1.66 (s, 3H; CH₃ -2β), 4.53 (s, 1H; CH-3), 5.22 and5.60 (2d, J=4 Hz, 2H; CH-5 and CH-6), and 5.82 (ABq, J=5.5 Hz, 2H; OCH₂O) ppm. Tetramethylsilane was used as internal reference.

EXAMPLE 9 Pivaloyloxymethyl 6β-iodopenicillanate A. Pivaloyloxymethyl6-diazopenicillanate

A stirred mixture of pivaloyloxymethyl 6β-aminopenicillanate (3.30 g, 10mmol) and sodium nitrite (1.38 g, 20 mmol) in methylene chloride (120ml) and water (120 ml) was cooled to 0° C. and treated with 2N aqueoussulphuric acid (7.5 ml) for 40 minutes. The organic phase was separated,dried (Na₂ SO₄), and concentrated at reduced pressure to about 30 ml.The concentrate was used immediately for the subsequent transformation.

B. Pivaloyloxymethyl 6α-iodopenicillanate

The concentrate of pivaloyloxymethyl 6-diazopenicillanate from step Aabove was diluted with acetone (120 ml), cooled to 0° C., and to thestirred mixture was added a cold solution of 67% aqueous hydroiodic acid(3.5 ml) and sodium iodide (4.5 g) in water (20 ml). After stirring fora furter 20 minutes at the low temperature, the mixture was treated withsolid sodium bicarbonate, filtered, and evaporated. The residue obtainedwas taken up in ethyl acetate (100 ml) and washed with 5% aqueous sodiumthiosulphate (2×75 ml). The organic phase was separated, dried (MgSO₄),and evaporated in vacuo. The residual oil was purified by columnchromatography on silica gel using ether-petroleum ether, 30:70, as theeluant to yield pure pivaloyloxymethyl 6α-iodopenicillanate ascolourless crystals from diisopropyl ether, melting point: 63°-64° C.

C. Pivaloyloxymethyl 6β-iodopenicillanate

To a stirred solution of pivaloyloxymethyl 6α-iodopenicillanate (0.88 g,2 mmol) in dry methylene chloride (20 ml) was added at -5° C. a 1Msolution of 1,5-diazabicyclo[4.3.0]non-5-ene (DBN) in dry methylenechloride (2 ml). The mixture was stirred at 0° C. for 20 minutes, shakenwith 1N aqueous acetic acid (2 ml), diluted with methylene chloride (20ml), washed with water (2×10 ml), dried (Na₂ SO₄), and evaporated invacuo to a dark oil. The residue was purified by column chromatographyon silica gel using ether-petroleum ether, 30:70, as the eluant toafford pivaloyloxymethyl 6β-iodopenicillanate as a slightly yellowishoil which crystallized from diisopropyl ether, melting point: 78°-79° C.

The NMR spectrum (CDCl₃) showed signals at δ=1.23 (s, 9H; C(CH₃)₃), 1.49(s, 3H; CH₃ -2α), 1.70 (s, 3H; CH₃ -2β), 4.55 (s, 1H; CH-3), 5.38 and5.62 (2d, J=4 Hz, 2H; CH-5 and CH-6), and 5.81 (ABq, J=5.5 Hz, 2H; OCH₂O) ppm. Tetramethylsilane was used as internal reference.

EXAMPLE 10 Acetoxymethyl 6β-bromopenicillanate

Chloromethyl acetate (0.11 ml, 1.2 mmol) was added to a solution ofpotassium 6β-bromopenicillanate (0.32 , 1 mmol) in dimethylformamide (5ml), and the mixtue was stirred for 16 hours at room temperature in adark room. After dilution with ether (20 ml), the mixture was washedwith water (4×5 ml), dried (MgSO₄), and evaporated in vacuo to yield thetitle compound as a yellowish oil.

The NMR spectrum (CDCl₃) showed signals at δ=1.49 (s, 3H; CH₃ -2α), 168(s, 3H; CH₃ -2β), 2.11 (s, 3H; COCH₃), 4.54 (s, 1H; CH-3), 5.33 and 5.59(2d, J=4 Hz, 2H; CH-5α and CH-6α), and 5.82 (ABq, J=5.5 Hz, 2H; OCH₂ O)ppm. Tetramethylsilane was used as internal reference.

EXAMPLE 11 Acetoxymethyl 6β-iodopenicillanate

Following the procedure of Example 10, but substituting potassium6β-iodopenicillanate for the potassium 6β-bromopenicillanate, the titlecompound was obtained as a yellowish oil.

The NMR spectrum (CDCl₃) showed signals at δ=1.50 (s, 3H; CH₃ -2α), 1.70(s, 3H; CH₃ -2β), 2.12 (s, 3H; COCH₃), 4.55 (s, 1H; CH-3), 5.39 and 5.63(2d, J=3.5 Hz, 2H; CH-5α and CH-6α), and 5.83 (ABq, J=5.5 Hz, 2H; OCH₂O) ppm. Tetramethylsilane was used as internal reference.

EXAMPLES 12-14 6β-Halopenicillanic acids

The crystalline 6β-halopenicillanic acids listed in Table III belowcould be obtained as follows:

(a) By concentrations at reduced pressure of the ethyl acetate solutionscontaining the pure 6β-halo compounds obtained after separation from thecorresponding 6α-epimers by dry column chromatography on silica gel (asdescribed in Example 1).

(b) By liberation from aqueous solutions of the corresponding potassiumsalts under a layer of ether or ethyl acetate at pH 3 followed byseparation of the organic phase, drying, and crystallization fromether-diisopropylether or ethyl acetate-hexane.

                  TABLE III    ______________________________________     ##STR4##                               .sup.1 HNMR data                  [α].sub.D.sup.20                               (δ/ppm; CD.sub.3 CN)    Example           X      (c = 0.5, CHCl.sub.3)                               C .sub.--H5 and C .sub.--H6    ______________________________________    12     Br     +272° 5.48 and 5.54, 2d, J = 4,0 Hz    13     Cl     +264° 5.38 and 5.58, 2d, J = 4.0 Hz    14     I      +276° 5.35 and 5.74, 2d, J = 4.0 Hz    ______________________________________

The above acids decompose at about 80°-100° C., therefore a well-definedmelting point cannot be determined.

EXAMPLE 15 Sodium 6β-bromopenicillanate A. Tetrabutylammonium6,6-dibromopenicillanate

To a solution of tetrabutylammonium hydrogen sulphate (34 g, 0.1 mol) inwater (50 ml), methylene chloride (100 ml) was added, followed by 2Nsodium hydroxide (50 ml). To the stirred mixture was added6,6-dibromopenicillanic acid (36 g, 0.1 mol) and the pH adjusted to 7.0with 2N sodium hydroxide. The organic layer was separated, and theaqueous phase was re-extracted with methylene chloride (50 ml). Afterdrying of the combined organic phases, ethyl acetate (500 ml) was added,and the solution was concentrated to about 300 ml in vacuo.

B. Sodium 6β-bromopenicillanate

To the above solution of tetrabutylammonium 6,6-dibromopenicillanate,tetrabutylammonium boranate (24.9 g, 0.1 mol) was added in one portionwith stirring. After about 30 minutes, the temperature in the mixturehad raised to 45°-50° C., whereafter it slowly decreased. After standingfor 1 hour, the solution was diluted with ether (300 ml), water (300 ml)was added, and the pH was adjusted to 3 with hydrochloric acid. Theorganic phase was separated and washed with water. Fresh water (50 ml)was added to the organic phase, and the pH was adjusted to 6.8 withaqueous potassium bicarbonate. The aqueous layer was separated, andwater was removed azeotropically with n-butanol in vacuo to yield acrystalline mixture of the potassium salts of 6β-bromo-, 6α-bromo-, and6,6-dibromopenicillanic acid in an approximate ratio of 65:25:10.

From an aqueous solution of the above salts, the free acids wereliberated at pH 3 (dilute hydrochloric acid) under a layer of ether, andthe resulting mixture was separated by column chromatography in asimilar way as described in Example 1 to yield, after salt formationwith 0.5M sodium bicarbonate and removal of water by azeotropicdistillation with n-butanol, crytalline sodium 6β-bromopenicillanate;[α]_(D) ²⁰ +266 ° (c=0.5, 1M phosphate buffer pH 7).

Calculated for C₈ H₉ BrNNaO₃ S: C, 31.80; H, 3.00; Br, 26.45; N, 4.64;S, 10.61%. Found: C, 31.85; H, 3.04; Br, 26.53; N, 4.56; S, 10.72%.

EXAMPLE 16 6β-Bromopenicillanic acid

To a stirred suspension of potassium 6,6-dibromopenicillanate (11.91 g,30 mmol) in dimethylformamide (30 ml) was added solution borohydride(1.14 g, 30 mmol). In the course of approximately 30 minutes, thetemperature in the reaction mixture rose to about 50° C., whereafter itslowly decreased to normal. After stirring at room temperature for 20hours, water (100 ml) and ether (100 ml) were added, and the pH of themixture was adjusted to 3 with dilute hydrochloric acid. The organiclayer was separated, the aqueous layer was extracted with ether (25 ml),and the combined organic extracts were washed with water (25 ml). To theorganic phase was added fresh water (25 ml), and the pH of the aqueousphase was adjusted to 7 by addition of 1M potassium bicarbonate withstirring. The aqueous layer was separated, and the water removedazeotropically by distillation with n-butanol in vacuo to give acrystalline mixture of the potassium salts of 6β- and6α-bromopenicillanic acid in an approximate ratio of 55:45, as indicatedby NMR spectroscopy.

The above potassium salts were dissolved in water (5 ml/g salt), and thepH of the aqueous phase was adjusted to 3 with 4N hydrochloric acidunder a layer of ethyl acetate 5 ml/g salt). The organic phase wasseparated, washed with water, dried, and diluted with an equal volume ofhexane. Seeding of the resulting solution followed by concentration atreduced pressure to about half the volume afforded crytalline6β-bromopenicillanic acid which was filtered off, washed with ethylacetate-hexane (1:1), and dried. Recrystallization fromether-diisopropyl ether gave the analytical sample, [α]_(D) ²⁰ +268°(c=0.5, CHCl₃).

Calculated for C₈ H₁₀ BrNO₃ S: C, 34.30; H, 3.60; Br, 28.53; N, 5.00; S,11.45%. Found: C, 34.47; H, 3.81; Br, 28.66; N, 4.99; S, 11.43%.

EXAMPLE 17 6β-Bromopenicillanic acid A. Dicyclohexylammonium6β-bromopenicillanate

To a solution of 6,6-dibromopenicillanic acid (10.8 g, 30 mmol) indimethylsulphoxide (75 ml) was added sodium cyanoborohydride (2.1 g; 90%pure), and the mixture was stirred until a clear solution was obtained(about 30 minutes). After standing for 72 hours, the mixture was dilutedwith water (75 ml) to precipitate unreacted starting material asdimethylsulphoxide solvate (C₈ H₉ Br₂ NO₃ S, C₂ H₆ OS). The crystalswere filtered off, washed with water and dried. The filtrate wasextracted with methylene chloride (4×25 ml), and the combined extractswere washed with water (50 ml), dried (Na₂ SO₄), and concentrated atreduced pressure to about half the volume. After addition ofdicyclohexylamine (2.5 ml) and acetone (50 ml), the mixture was furtherconcentrated to about 25 ml. Crystallization was induced by scratching,and, after standing for 1 hour at room temperature, the puredicyclohexylammonium 6β-bromopenicillanate was filtered off, washed withacetone, and dried. The compound exhibited no well-defined meltingpoint, after darkening at about 170° C., it decomposed at 280°-290° C.

B. 6β-Bromopenicillanic acid

A stirred suspension of dicyclohexylammonium 6β-bromopenicillanate inethylacetate-water (1:1) (20 ml/g salt) was adjusted to pH 3 with 4Nhydrochloric acid. Precipitated dicyclohexylammonium chloride wasfiltered off, and the organic layer was separated, washed twice withwater, and dried. Addition of an equal volume of hexane followed byconcentration of the solution at reduced pressure yielded pure,crystalline 6β-bromopenicillanic acid, identical with the compounddescribed in Examples 12 and 16.

EXAMPLE 18 Sodium 6β-iodopenicillanate A. 6,6-Diiodopenicillanic acidmorpholine salt

To a stirred solution of 6-aminopenicillanic acid (110 g, ˜0.5 mol) in amixture of 5N sulphuric acid (400 ml) and methylene chloride (1.5 liter)were added dropwise and simultaneously at 0° C. 2.5M aqueous sodiumnitrite (340 ml) and 0.5M methanolic iodine (1 liter). After theaddition was finished, the cooling bath was removed, and stirring of themixture was continued for 1 hour. The organic layer was separated, andthe aqueous phase was extracted with methylene chloride (200 ml). Thecombined organic extracts were washed with 1M aqueous sodiumthiosulphate (600 ml) and dried (Na₂ SO₄). After addition of morpholine(32.6 ml, 0.375 mol), the resulting solution was concentrated at reducedpressure to about 200-250 ml to afford, after cooling, the titlecompound as colorless crystals which were collected, washed withacetone, and dried. Yield: 162.6 g; melting point: 152°-154° C.(decomposition).

Calculated for C₁₂ H₁₈ I₂ N₂ O₄ S: C, 26.68; H, 3.36; I, 46.99; N, 5.19;S, 5.94%. Found: C, 27.01; H, 3.44; I, 46.70; N, 5.18; S, 5.64%.

B. Sodium 6β-iodopenicillanate

A stirred solution of 6,6-diiodopenicillanic acid morpholine salt (54 g,0.1 mol) in methylene chloride (500 ml) was protected from light, andcetyl trimethylammonium boranate (36 g 0.12 mol) was added. Afterstirring for 15 minutes at room temperature, the mixture was evaporatedin vacuo. The residue was triturated with acetone (250 ml), insolublesalt was removed by filtration and the filtrate evaporated to dryness.The residual oil was dissolved in ethyl acetate (200 ml), water (200 ml)was added, and the pH in the aqueous phase was adjusted to 7 by additionof 2N sodium hydroxide with stirring. The aqueous layer was separated,the organic phase washed with water (50 ml), and the pH of the combinedaqueous phases was adjusted to 3 with dilute hydrochloric acid under alayer of ether (200 ml). The organic phase was separated, the aqueousphase was re-extracted with ether, and the combined ethereal extractswere dried and concentrated at reduced pressure to about 80-100 ml. Theconcentrate contained a mixture of 6β- and 6α-iodopenicillanic acids aswell as minor amounts of penicillanic acid (approximate ratio 50:40:10)which were separated by dry column chromatography using a similarprocedure as described in Example 1. The pure 6β-iodo acid thus obtainedgave, after salt formation with 0.5M aqueous sodium bicarbonate andremoval of water by azeotropic distillation with n-butanol, crystallinesodium 6β-iodopenicillanate; [α]_(D) ²⁰ +274° (c=0.5, 1M phosphatebuffer pH 7).

Calculated for C₈ H₉ INNaO₃ S: C, 27.52; H, 2.60; I, 36.35; N, 4.01; S,9.18%. Found: C, 27.31; H, 2.64; I, 36.12; N, 3.92; S, 9.34%.

EXAMPLE 19 6β-Iodopenicillanic acid A. 6,6-Diiodopenicillanic aciddimethylsulphoxide solvate

To a cooled solution of 6,6-diiodopenicillanic acid morpholine salt(10.8 g, 20 mmol) in dimethylsulphoxide (20 ml) was added 1Nhydrochloric acid (20 ml), and crystallization was induced byscratching. After further addition of water (20 ml), the crystals werefiltered off, washed with water, and dried to give an almostquantitative yield of the title compound which showed an ill-definedmelting point with slow decomposition aove 120°-125° C.

Calculated for C₈ H₉ I₂ NO₃ S, C₂ H₆ OS: C, 22.61; H, 2.85; I, 47.78; N,2.64; S, 12.07%. Found: C, 22.96; H, 2.81; I, 47.64; N, 2.74; S, 12.14%.

B. Dicyclohexylammonium 6β-iodopenicillanate

To a solution of 6,6-diiodopenicillanic acid dimethylsulphoxide solvate(5.31 g, 10 mmol) in dimethylsulphoxide (25 ml) was added sodiumcyanoborohydride (0.7 g; 90% pure), and the mixture was stirred until aclear solution was obtained (about 30 minutes). After standing for 40hours at room temperature, water (50 ml) was added, and the mixture wascooled to 5° C. to precipitate unreacted starting material which wascollected, washed with water, and dried. The filtrate was extracted withmethylene chloride (3×25 ml), and the combined extracts were washed withwater (2×10 ml), dried (Na₂ SO₄), and carefully evaporated in vacuo. Theresidual oil was dissolved in acetone (25 ml), an equivalent amount ofdicyclohexylamine was added, and crystallization was induced byscratching. After standing for 1 hour, the pure dicyclohexylammonium6β-iodopenicillanate was filtered off, washed with acetone, and dried.The compound showed no well-defined melting point, after darkening atabout 150° C., it decomposed slowly above this temperature.

C. 6β-Iodopenicillanic acid

By substituting dicyclohexylammonium 6β-iodopenicillanate for thecorresponding 6β-bromopenicillanate in the procedure of Example 17B,6β-iodopenicillanic acid was obtained as colorless crystals.Recrystallization from ether-diisospropyl ether afforded the analyticalsample, [α]_(D) ²⁰ +278° (c=0.5, CHCl₃).

Calculated for C₈ H₁₀ INO₃ S: C, 29.37; H, 3.08; I, 38.79; N, 4,28; S,9.80%. Found: C, 29.46; H, 3.13; I, 38.96; N, 4.27; S, 9.81%.

EXAMPLE 20 Pivaloyloxymethyl 6β-bromopenicillanate A. Pivaloyloxymethyl6,6-dibromopenicillanate

To a solution of potassium 6,6-dibromopenicillanate (5.96 g, 15 mmol) indimethylformamide (30 ml) was added chloromethyl pivalate (2.22 ml, 15mmol), and the mixture was stirred for 16 hours at room temperature.After dilution with ethyl acetate (120 ml), the mixture was washed withwater (4×30 ml), dried, decolorized by stirring with charcoal (0.5 g; 1hour), and evaporated to dryness to give the desired compound as ayellow oil which crystallized from ether-hexane; melting point:101°-102° C.

B. Pivaloyloxymethyl 6β-bromopenicillanate

To a solution of pivaloyloxymethyl 6,6-dibromopenicillanate (5.68 g, 12mmol) in dimethylsulphoxide (25 ml) was added sodium cyanoborohydride(0.84 g; 90% pure), and the mixture was stirred for 24 hours at roomtemperature in a dark room. After addition of water (75 ml), the mixturewas extracted with ether (3×25 ml), and the combined ethereal extractswere washed with water (3×10 ml), dried, and concentrated at reducedpressure to about 20 ml. The concentrate was subjected to columnchromatography on silica gel similar to the procedure described inExample 9C. Hereby, the 6β-bromo compound was separated from unreactedstarting material. Fractions containing the more polar 6β-bromo esterwere combined and evaporated in vacuo. The residual oil was crystallizedfrom ether-diisopropyl ether to give pivaloyloxymethyl6β-bromopenicillanate, melting point: 66°-68° C., identical with thecompound described in Example 9C.

EXAMPLE 21 6β-Bromopenicillanic acid pivampicillin salt

To a stirred solution of pivampicillin hydrochloride (2.50 g, 5 mmol) inwater (100 ml) was added dropwise 0.1M aqueous potassium6β-bromopenicillanate (50 ml). The colorless precipitate thus obtainedwas filtered off, washed with water (3×10 ml), and dried in vacuo togive the pure title compound as colorless crystals which began todecompose at 120°-130° C. without melting.

The IR-spectrum (KBr) showed bands at 3030, 2970, 2935, 2870, 1790,1770, 1680, 1600, and 627 cm⁻¹.

The NMR-spectrum (CDCl₃) showed signals at δ=1.20 (s, 9H; C(CH₃)₃), 1.36(s, 3H; CH₃ -2), 1.44 (s, 3H; CH₃ -2), 1.52 (s, 3H; CH₃ -2), 1.54 (s,3H; CH₃ -2), 4.26 (s, 1H; CH-3), 4.40 (s, 1H; CH-3), 5.11 (s, 1H; CHCO),5.23, 5.36, 5.43, and ˜5.76 (4 doublets, J=3.8-4.2 Hz, 4H; CH-5 andCH-6), 5.79 (ABq, J=5.5 Hz, 2H; OCH₂ O), 7.40 (s, 5H; arom. CH), and7.82 (d, J=8.2 Hz, 1H; CONH) ppm. Tetramethylsilane was used as internalreference.

EXAMPLE 22 6β-Iodopenicillanic acid bacampicillin salt

A solution of potassium 6β-iodopenicillanate (0.73 g, 2 mmol) in water(20 ml) was added dropwise to a stirred solution of bacampicillinhydrochloride (1.0 g, 2 mmol) in water (40 ml). The resultingcrystalline precipitate was filtered off, washed with water, and driedto afford the pure title compound which decomposed at 110°-120° C.without melting.

The IR-spectrum (KBr) showed bands at 3030, 2980, 2870, 1780, 1765,1695, 1625, and 618 cm⁻¹.

The NMR-spectrum (CDCl₃) showed signals at δ=1.31 (t, J=7 Hz, 3H; OCH₂CH₃), 1.38 (s, 3H; CH₃ -2), 1.49 (s, 3H; CH₃ -2), 1.54 (s, 3H; CH₃ -2),1.59 (s, 3H; CH₃ -2), 4.25 (m, 4H; OCH₂ CH₃ and CH-3), 4.99 (s, 1H;CHCO), 5.12 (bs, NH₃ ⁺), 5.21, 5.45, 5.54, and 5.64 (4 doublets,J=3.8-4.2 Hz, 4H; CH-5 and CH-6), 6.76 (ABq, J=5.5 Hz, 1H; CHCH₃), 7.39(s, 5H; arom. CH), and 7.72 (d, J=8.5 Hz, 1H; CONH) ppm.Tetramethylsilane was used as internal reference.

EXAMPLES 23-25 Further salts of 6β-halopenicillanic acids with inorganicbases

Treatment of an ethereal solution of the 6β-halopenicillanic acid withan equivalent amount of aqueous base followed by separation of theaqueous phase and freeze-drying afforded the salts listed in Table IV ascolorless powders.

                  TABLE IV    ______________________________________     ##STR5##    Example        X     n           Z    ______________________________________    23a            Cl    1           Na.sup.+    23b            Cl    2           Ca.sup.++    24a            Br    1           Li.sup.+    24b            Br    2           Ca.sup.++    25a            I     1           Li.sup.+    25b            I     2           Ca.sup.++    ______________________________________

EXAMPLES 26-28 Further salts of 6β-halopenicillanic acids with organicbases

By treatment of a solution of the 6β-halopenicillanic acid in a suitableorganic solvent, e.g. acetone, ethyl acetate or ether, with anequivalent amount of the organic base (preferably dissolved in the samesolvent), the desired salt was obtained as a crystalline precipitatewhich was filtered off and dried in vacuo. The salts obtained by thismethod are listed in Table V below.

                  TABLE V    ______________________________________     ##STR6##    Example   X     n       Z    ______________________________________    26a       Cl    1       dicyclohexylamine    26b       Cl    2       N,N'dibenzylethylenediamine    27a       Br    1       morpholine    27b       Br    2       N,N'dibenzylethylenediamine    27c       Br    1       Nethylpiperidine    27d       Br    1       procaine    28a       I     1       dibenzylamine    28b       I     2       N,N'dibenzylethylenediamine    28c       I     1       Nmethylbenzylamine    28d       I     1       procaine    ______________________________________

EXAMPLES 29-30 Salts of 6β-halopenicillanic acids with β-lactamantibiotics and pro-drugs thereof containing a basic group

The salts listed in Table VI below were prepared by procedures similarto those described in the preceeding Examples 21-22(A), 23-25(B), or26-28(C).

                  TABLE VI    ______________________________________     ##STR7##    Ex-                    Pro-    am-                    ce-    ple  X     Z           dure IR-data (cm.sup.-1)    ______________________________________    29a  Br    Bacampicillin                           A    3010, 2980, 2940, 1785, 1765,                                1690, 1620, 628    29b  Br    Pivmecillinam                           C    2970, 2932, 2865, 1775, 1690,                                1630, 1600, 625    29c  Br    Bacmecillinam                           C    2970, 2935, 2860, 1770, 1686,                                1630, 1600, 628    29d  Br    Penethamate C    3455, 3020, 2940, 2860, 2660,                                1800, 1780, 1745, 1675, 625    29e  Br    Ampicillin  B    29f  Br    Amoxycillin B    30a  I     Pivampicillin                           A    3040, 2975, 2935, 2870, 1782,                                1770, 1685, 1600, 618    30b  I     Pivmecillinam                           C    2970, 2935, 2865, 1780, 1770,                                1685, 1630, 1600, 616    30c  I     Bacmecillinam                           C    2970, 2930, 2860, 1775, 1760,                                1685, 1630, 1600, 616    30d  I     Penethamate C    3460, 3018, 2940, 2855, 2665                                1795, 1775, 1745, 1670, 615    30e  I     Ampicillin  B    30f  I     Amoxycillin B    ______________________________________

EXAMPLE 31 Tablets

    ______________________________________    Component               Per tablet    ______________________________________    6β-Iodopenicillanic acid sodium salt                            250    mg    Microcrystalline cellulose                            110    mg    Hydroxypropyl cellulose 5      mg    Alginic acid            10     mg    Talc                    23     mg    Magnesium stearate      2      mg                            400    mg    ______________________________________

The active component is blended with the microcrystalline cellulose,granulated with a 10% solution of hydroxypropyl cellulose inisopropanol, dried at 40° C., and screened through 1 mm sieves. Alginicacid, talc, and magnesium stearate are added, and the mixture iscompressed into tablets each weighing 400 mg. The tablets are coveredwith a film-coating of hydroxypropyl methyl cellulose.

EXAMPLE 32 Tablets

    ______________________________________    Component                Per tablet    ______________________________________    6β-Bromopenicillanic acid sodium salt                             250    mg    Microcrystalline cellulose                             110    mg    Hydroxypropyl cellulose  5      mg    Alginic acid             10     mg    Talc                     23     mg    Magnesium stearate       2      mg                             400    mg    ______________________________________

By the same method as described in Example 31, the tablets containing6β-bromopenicillanic acid sodium salt are obtained.

EXAMPLE 33 Parenteral formulation

    ______________________________________    Component             Per vial    ______________________________________    6β-Iodopenicillanic acid sodium salt                          125 mg    ______________________________________

The sterile crystalline component is filled into sterile vials which areaseptically sealed. For parenteral administration, 2 ml of sterilephysiological saline is added to the content of the vial.

EXAMPLE 34 Parenteral formulation

    ______________________________________    Component              Per vial    ______________________________________    6β-Bromopenicillanic acid sodium salt                           125 mg    ______________________________________

The above formulation is obtained by the same method as described inExample 33.

EXAMPLE 35 Capsules

    ______________________________________    Component                Per capsule    ______________________________________    6β-Iodopenicillanic acid potassium salt                             125    mg    Hydroxypropyl cellulose  3      mg    Talc                     6      mg    Magnesium stearate       1      mg                             135    mg    ______________________________________

The active ingredient is granulated with a solution of hydroxypropylcellulose in isopropanol, dried at 40° C., and screened through 1 mmsieves. Talc and magnesium stearate are added, the components are wellmixed to obtain a uniform blend, and 135 mg of the blend is filled in aNo. 3 hard gelatine capsule.

EXAMPLE 36 Capsules

    ______________________________________    Component               Per capsule    ______________________________________    6β-Bromopenicillanic acid pivampi-                            250    mg    cillin salt    Hydroxypropyl cellulose 5      mg    Talc                    23     mg    Magnesium stearate      2      mg                            280    mg    ______________________________________

The active compound is granulated with a solution of hydroxypropylcellulose in isopropanol, dried at 40° C., and screened through 1 mmsieves. The diluents are added, and the mixture is carefully blended,280 mg of the blend being filled in No. 2 gelatine capsules.

EXAMPLE 37 Tablets

    ______________________________________    Component               Per tablet    ______________________________________    6β-Iodopenicillanic acid sodium salt                            125    mg    Ampicillin              250    mg    Corn starch             75     mg    Hydroxypropyl cellulose 10     mg    Alginic acid            10     mg    Talc                    20     mg    Magnesium stearate      5      mg                            495    mg    ______________________________________

In the above composition, the active components are blended with cornstarch, granulated with a 10% solution of hydroxypropyl cellulose inisopropanol, dried at 40° C., and screened through 1 mm screens. Alginicacid, talc, and magnesium stearate are added, and the mixture iscompressed into tablets each weighing 495 mg. The tablets are coveredwith a film-coating of hydroxypropyl methyl cellulose.

EXAMPLE 38 Tablets

    ______________________________________    Component                Per tablet    ______________________________________    6β-Bromopenicillanic acid potassium salt                             125    mg    Ampicillin               250    mg    Corn starch              75     mg    Hydroxypropyl cellulose  10     mg    Alginic acid             10     mg    Talc                     20     mg    Magnesium stearate       5      mg                             495    mg    ______________________________________

By the same method as described in Example 37, the tablets containing6β-bromopenicillanic acid potassium salt and ampicillin are obtained.

EXAMPLE 39 Tablets

    ______________________________________    Component                Per tablet    ______________________________________    6β-Iodopenicillanic acid potassium salt                             200    mg    Pivampicillin free base  250    mg    Corn starch              100    mg    Hydroxypropyl cellulose  5      mg    Methyl cellulose         5      mg    Alginic acid             15     mg    Talc                     20     mg    Magnesium stearate       5      mg                             600    mg    ______________________________________

In the above composition, 6β-iodopenicillanic acid potassium salt andhalf the amount of corn starch are blended, granulated with a 10%solution of hydroxypropyl cellulose in isopropanol, dried at 40° C. andscreened through 1 mm screens. The balance of corn starch is blendedwith pivampicillin, granulated with paste of methyl cellulose and water,dried at 50° C., and screened through 0.7 mm screens. The granulates areblended with alginic acid, talc, and magnesium stearate, and the mixtureis compressed into tablets each weighing 600 mg. The tablets are coveredwith a film-coating of hydroxypropyl methyl cellulose.

EXAMPLE 40 Tablets

    ______________________________________    Component               Per tablet    ______________________________________    6β-Bromopenicillanic acid sodium salt                            150    mg    Pivampicillin free base 250    mg    Corn starch             100    mg    Hydroxypropyl cellulose 5      mg    Methyl cellulose        5      mg    Alginic acid            15     mg    Talc                    20     mg    Magnesium stearate      5      mg                            550    mg    ______________________________________

The tablets containing 6β-bromopenicillanic acid sodium salt andpivampicillin free base are obtained by the same method as described inExample 39.

EXAMPLE 41 Tablets

    ______________________________________    Component               Per tablet    ______________________________________    6β-Iodopenicillanic acid sodium salt                            125    mg    Amoxycillin             250    mg    Corn starch             80     mg    Hydroxypropyl cellulose 10     mg    Alginic acid            10     mg    Talc                    20     mg    Magnesium stearate      5      mg    ______________________________________

The active ingredients are blended with the corn starch, granulated witha 10% solution of hydroxypropyl cellulose in isopropanol, dried at 40°C., and screened through 1 mm screens. After addition of alginic acid,talc, and magnesium stearate, the mixture is compressed into tabletseach weighing 500 mg. The tablets are covered with a film-coating ofhydroxypropyl methyl cellulose.

EXAMPLE 42 Tablets

    ______________________________________    Component                Per tablet    ______________________________________    6β-Bromopenicillanic acid potassium salt                             125    mg    Amoxycillin              250    mg    Corn starch              80     mg    Hydroxypropyl cellulose  10     mg    Alginic acid             10     mg    Talc                     20     mg    Magnesium stearate       5      mg                             500    mg    ______________________________________

By the same method as described in Example 41, the tablets containing6β-bromopenicillanic acid potassium salt and amoxycillin are obtained.

EXAMPLE 43 Tablets

    ______________________________________    Component                Per tablet    ______________________________________    6β-Iodopenicillanic acid pivampicillin salt                             500    mg    Hydroxypropyl cellulose  10     mg    Microcrystalline cellulose                             200    mg    Magnesium stearate       10     mg                             710    mg    ______________________________________

The active component is granulated with a 15% solution of hydroxypropylcellulose and isopropanol, dried at 40° C., and screened through 1 mmsieves. Microcrystalline cellulose and magnesium stearate are added, andthe mixture is compressed into tablets each weighing 710 mg.

EXAMPLE 44 Tablets

    ______________________________________    Component                Per tablet    ______________________________________    6β-Bromopenicillanic acid pivmecillinam                             350    mg    salt    Hydroxypropyl cellulose  10     mg    Microcrystalline cellulose                             130    mg    Magnesium stearate       10     mg                             500    mg    ______________________________________

The tablets containing the pivmecillinam salt of 6β-bromopenicillanicacid are obtained by the same method as described in Example 43.

EXAMPLE 45 Tablets

    ______________________________________    Component                Per tablet    ______________________________________    6β-Iodopenicillanic acid potassium salt                             125    mg    Cephalexin               250    mg    Corn starch              80     mg    Hydroxypropyl cellulose  10     mg    Alginic acid             10     mg    Talc                     20     mg    Magnesium stearate       5      mg                             500    mg    ______________________________________

The tablets containing 6β-iodopenicillanic acid potassium salt andCephalexin are obtained by the same method as described in Example 41.

EXAMPLE 46 Tablets

    ______________________________________    Component                Per tablet    ______________________________________    6β-Bromopenicillanic acid sodium salt                             125    mg    Cefaclor                 250    mg    Corn starch              80     mg    Hydroxypropyl cellulose  10     mg    Alginic acid             10     mg    Talc                     20     mg    Magnesium stearate       5      mg                             500    mg    ______________________________________

By the same method as described in Example 41, the tablets containing6β-bromopenicillanic acid sodium salt and Cefaclor are obtained.

EXAMPLE 47 6β-Iodpenicillanic acid sodium salt/Pivmecillinam HClTwo-layer tablets

    ______________________________________    Granulate I    Component               Per tablet    ______________________________________    6β-Iodopenicillanic acid sodium salt                            100    mg    Hydroxypropyl cellulose 2      mg    Dicalcium phosphate dihydrate                            86     mg    Sodium starch glycolate 10     mg    Magnesium stearate      2      mg    ______________________________________

The 6β-iodopenicillanic acid sodium salt is granulated with a solutionof hydroxypropyl cellulose in isopropanol, dried at 40° C., and screenedthrough 0.75 mm screens. The granules are blended with dicalciumphosphate dihydrate, sodium starch glycolate and magnesium stearate.

    ______________________________________    Granulate II    Component              Per tablet    ______________________________________    Pivmecillinam hydrochloride                           100    mg    Hydroxypropyl cellulose                           2      mg    Microcrystalline cellulose                           50     mg    Magnesium stearate     1.5    mg    Talc                   3.5    mg    ______________________________________

Pivmecillinam hydrochloride is granulated with a solution ofhydroxypropyl cellulose, dried at 40° C., and screened through 0.75 mmscreens. The granules are blended with microcrystalline cellulose,magnesium stearate, and talc.

The granulates are compressed into two-layer tablets containing 200 mgof granulate I as the bottom layer and 157 mg of granulate II as the toplayer.

The tablets are film-coated with hydroxypropylmethyl cellulose dissolvedin ethanol-water (1:1).

EXAMPLE 48 6β-Bromopenicillanic acid sodium salt/Pivampicillin baseTwo-layer tablets

    ______________________________________    Granulate I    Component                Per tablet    ______________________________________    6β-Bromopenicillanic acid sodium salt                             100    mg    Polyvinyl pyrrolidone    5      mg    Dicalcium phosphate dihydrate                             85     mg    Sodium starch glycolate  8      mg    Magnesium stearate       2      mg    ______________________________________

The 6β-bromopenicillanic acid sodium salt is granulated with a solutionof polyvinyl pyrrolidone in isopropanol, dried at 40° C., screenedthrough 0.75 mm screens, and blended with dicalcium phosphate dihydrate,starch, and magnesium stearate.

    ______________________________________    Granulate II    Component              Per tablet    ______________________________________    Pivampicillin base     125       mg    Starch                 30        mg    Hydroxypropyl cellulose                           3.5       mg    Magnesium stearate     1.5       mg    ______________________________________

The pivampicillin base and 10 mg of the starch are blended andgranulated with a solution of hydroxypropyl cellulose in de-ionizedwater, dried at 50° C., and screened through 0.75 mm screens. Thegranules are blended with the rest of the starch and magnesium stearate.

The granulates are compressed into tablets containing 200 mg ofgranulate I as the bottom layer and 160 mg of granulate II as the toplayer. The tablets are film-coated with hydroxypropylmethyl cellulosedissolved in de-ionized water-ethanol (1:1).

EXAMPLE 49 6β-Bromopenicillanic acid sodium salt/Amoxycillin Two-Layertablets

    ______________________________________    Granulate I    Component                Per tablet    ______________________________________    6β-Bromopenicillanic acid sodium salt                             100    mg    Polyvinyl pyrrolidone    7      mg    Lactose                  50     mg    Silicium dioxide         1.5    mg    Magnesium stearate       1.5    mg    ______________________________________

The 6β-bromopenicillanic acid sodium salt is granulated with a solutionof polyvinyl pyrrolidone in isopropanol, dried at 40° C., and screenedthrough 0.75 mm screens. The granules are blended with lactose, siliciumdioxide and magnesium stearate.

    ______________________________________    Granulate II    Component             Per tablet    ______________________________________    Amoxycillin           200    mg    Starch                30     mg    Polyvinyl pyrrolidone 6      mg    Silicium dioxide      2      mg    Magnesium stearate    2      mg    ______________________________________

Amoxycillin is blended with starch, granulated with a solution ofpolyvinyl pyrrolidone in de-ionized water, dried at 50° C., and screenedthrough 0.75 mm screens. The granules are blended with silicium dioxideand magnesium.

The granulates are compressed into tablets containing 240 mg ofgranulate II as the bottom layer and 160 mg of granulate I as the toplayer.

EXAMPLE 50 6β-Iodopenicillanic acid sodium salt/PivmecillinamHCl/Pivampicillin Three-layer tablets

    ______________________________________    Granulate I    Component               Per tablet    ______________________________________    6β-Iodopenicillanic acid sodium salt                            100    mg    Hydroxypropyl cellulose 2      mg    Microcrystalline cellulose                            50     mg    Sodium starch glycolate 6      mg    Magnesium stearate      2      mg    ______________________________________

6β-Iodopenicillanic acid sodium salt is granulated with a solution ofhydroxypropyl cellulose in isopropanol, dried at 40° C., and screenedthrough 0.75 mm screens, whereafter microcrystalline cellulose, sodiumstarch glycolate, and magnesium stearate are added.

    ______________________________________    Granulate II    Component              Per tablet    ______________________________________    Pivmecillinam hydrochloride                           100    mg    Hydroxypropyl cellulose                           2      mg    Microcrystalline cellulose                           50     mg    Magnesium stearate     1.5    mg    ______________________________________

Pivmecillinam hydrochloride is granulated with a solution ofhydroxypropyl cellulose in isopropanol, dried at 40° C., and screenedthrough 0.75 mm screens. The granules are blended with microcrystallinecellulose and magnesium stearate.

    ______________________________________    Granulate III    Component              Per tablet    ______________________________________    Pivampicillin base     125    mg    Starch                 24     mg    Hydroxypropyl cellulose                           3.5    mg    Sodium starch glycolate                           7      mg    Magnesium stearate     1.5    mg    ______________________________________

The pivampicillin base and 10 mg starch are blended and granulated witha solution of hydroxypropyl cellulose dissolved in de-ionized water, andgranulated mass being dried at 50° C., and screened through 0.75 mmscreens. Thereafter sodium starch glycolate, the rest of the starch, andmagnesium stearate are added.

The granulates are compressed into tablets containing 160 mg ofgranulate I as the bottom layer, 153.5 mg of granulate II as theintermediate layer, and 161 mg of granulate III as the top layer. Thetablets are covered with a film-coating of hydroxypropylmethyl cellulosedissolved in de-ionized water-ethanol (1:1).

EXAMPLE 51

The following composition is prepared:

    ______________________________________    Penethamate hydroiodide   100    g    Potassium salt of         100    g    6-β-bromo penicillanic acid    12-hydroxystearin, known under the                              20     g    trade name of "Thixin R"    Modified coconut oil, known under the                              780    g    trade names of "Miglyol 812R" and    "Neobee R"                              1000   g    ______________________________________

The 12-hydroxystearin is dissolved in the coconut oil at 70° C. andcooled to room temperature. Penethamate hydroiodide and the potassiumsalt of 6-β-bromo penicillanic acid are incorporated therein byagitation followed by homogenization. The resulting suspension is filledinto plastic syringes, each containing 5 g of the suspension.

EXAMPLE 52

The following composition is prepared in accordance with the proceduresset forth in Example 51.

    ______________________________________    Penethamate hydroiodide 100    g    Potassium salt of       50     g    6-β-iodo penicillanic acid    Polysorbate 80          10     g    12-hydroxystearin       25     g    Modified coconut oil    815    g                            1000   g    ______________________________________

The resulting suspension was then filled into plastic syringes, eachcontaining 5 g of the suspension.

EXAMPLE 53

The following composition is prepared

    ______________________________________    Penethamate hydroiodide 20     g    Potassium salt of       40     g    6-β-bromo penicillanic acid    Framycetin sulfate      20     g    Aluminum monostearate   20     g    12-hydroxystearin       10     g    Liquid paraffin         890    g                            1000   g    ______________________________________

The aluminum monostearate and the 12-hydroxystearin are dissolved in theliquid paraffin at 130° C. and cooled to 30° C. The penethamatehydroiodide, potassium salt of 6-β-iodo penicillanic acid and framycetinsulfate are incorporated therein by agitation followed byhomogenization. The resulting suspension is filled into plasticsyringes, each containing 5 g of the suspension.

EXAMPLE 54

The procedures of Example 51 were repeated to prepare a similarcomposition except that the penethamate hydroiodide was replaced by anequivalent amount of the salt prepared in Example 29d. 195 syringes ofthe resulting suspension were prepared.

What we claim is:
 1. A process for the preparation of an essentiallypure compound for the formula I: ##STR8## or salts thereof, wherein Xstands for chlorine, bromine or iodine, which process comprisesbase-catalyzed epimerization of a salt of a 6α-halopenicillanic acid inaqueous solution or stereoselective reduction of a6,6-dihalopenicillanic acid or a salt thereof with alkali metal ortetraalkylammonium borohydride or cyanoborohydride, followed by recoveryof the pure compound of formula I or a salt thereof by chromatographicseparation or fractionate crystallization.
 2. A process as claimed inclaim 1, wherein the epimerization is carried out at a pH of 9.0 to 9.2and at a temperature of 30° C. to 32° C. for a period of 20 to 24 hours,the pH being kept constant during the course of the reaction by additionof dilute aqueous sodium or potassium hydroxide via an automatictitrator.
 3. A process as claimed in claim 2, wherein the reduction iscarried out in a polar organic solvent selected from the groupconsisting of dimethylformamide and dimethyl sulfoxide.
 4. A process asclaimed in claim 1, wherein the pure compound of formula I is recoveredin the form of a dicyclohexylammonium salt.